The development of nucleic acid amplification technology has revolutionized genetic analysis and engineering science. For example, the polymerase chain reaction (PCR) is commonly utilized to amplify specific target nucleic acids using selected primer nucleic acids, e.g., to facilitate the detection of the target nucleic acid as part of a diagnostic, forensic, or other application. Primers typically function in pairs that are designed for extension towards each other to cover the selected target region. A typical PCR cycle includes a high temperature (e.g., 85° C. or more) denaturation step during which the strands of double-stranded nucleic acids separate from one another, a low temperature (e.g., 45-65° C.) annealing step during which the primers hybridize to the separated single strands, and an intermediate temperature (e.g., around 72° C.) extension step during which a nucleic acid polymerase extends the primers. Two-temperature thermocycling procedures are also utilized. These generally include a high temperature denaturation step and a low temperature anneal-extend step.
PCRs are also described in many different U.S. patents including, e.g., U.S. Pat. No. 4,683,195, entitled “PROCESS FOR AMPLIFYING, DETECTING, AND/OR-CLONING NUCLEIC ACID SEQUENCES,” which issued to Mullis et al. Jul. 28, 1987, U.S. Pat. No. 4,683,202, entitled “PROCESS FOR AMPLIFYING NUCLEIC ACID SEQUENCES,” which issued to Mullis Jul. 28, 1987, and U.S. Pat. No. 4,965,188, entitled “PROCESS FOR AMPLIFYING, DETECTING, AND/OR CLONING NUCLEIC ACID SEQUENCES USING A THERMOSTABLE ENZYME,” which issued to Mullis et al. Oct. 23, 1990, which are each incorporated by reference. Further, PCR-related techniques are also described in various other publications, such as Innis et al. (Eds.) PCR Protocols: A Guide to Methods and Applications, Elsevier Science & Technology Books (1990), Innis et al. (Eds.) PCR Applications: Protocols for Functional Genomics, Academic Press (1999), Edwards et al., Real-Time PCR, Taylor & Francis, Inc. (2004), and Rapley et al., Molecular Analysis and Genome Discovery, John Wiley & Sons, Inc. (2004), which are each incorporated by reference.
Many variations of the PCR as well as other nucleic acid amplification techniques have also been developed. Examples of these include reverse-transcription PCR (RT-PCR) (Joyce (2002) “Quantitative RT-PCR. A review of current methodologies” Methods Mol. Biol. 193:83-92 and Emrich et al. (2002) “Quantitative detection of telomerase components by real-time, online RT-PCR analysis with the LightCycler,” Methods Mol. Biol. 191:99-108), the ligase chain reaction (LCR) (Lee (1996) “Ligase chain reaction,” Biologicals 24(3):197-9), the polymerase ligase chain reaction (Barany et al. (1991) “The ligase chain reaction in a PCR world,” PCR Methods Appl. 1(1):5-16), the Gap-LCR (Abravaya et al. (1995) “Detection of point mutations with a modified ligase chain reaction (Gap-LCR),” Nucleic Acids Res. 23(4):675-82), strand displacement amplification (Walker (1993) “Empirical aspects of strand displacement amplification,” PCR Methods Appl. 3(1):1-6), linked linear amplification (LLA) (Killeen et al. (2003) “Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations,” Clin Chem. 49(7):1050-7), rolling circle amplification (RCA) (Nilsson et al. (2002) “Real-time monitoring of rolling-circle amplification using a modified molecular beacon design,” Nucleic Acids Res. 30(14):e66), transcription-mediated amplification (TMA) (Emery et al. (2000) “Evaluation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay using primary subtype A, C, and D isolates from Kenya,” J Clin Microbiol 38:2688-2695), nucleic-acid-sequence-based amplification (NASBA) (Mani et al. (1999) “Plasma RNA viral load as measured by the branched DNA and nucleic acid sequence-based amplification assays of HIV-1,” J Acquir Immune Defic Syndr 22:208-209 and Berndt et al. (2000) “Comparison between a nucleic acid sequence-based amplification and branched DNA test for quantifying HIV RNA load in blood plasma,” J Virol Methods 89:177-181), and self-sustaining sequence replication (3SR) (Mueller et al. (1997) “Self-sustained sequence replication (3SR): an alternative to PCR,” Histochem Cell Biol 108:431-7), which are each incorporated by reference.
Various strategies for detecting amplification products have been developed, including those involving 5′ nuclease probes, molecular beacons, or SCORPION® primers, among many others. To illustrate, a 5′ nuclease assay typically utilizes the 5′ to 3′ nuclease activity of certain DNA polymerases to cleave 5′ nuclease probes during the course of a polymerase chain reaction (PCR). These assays allow for both the amplification of a target and the release of labels for detection, generally without resort to multiple handling steps of amplified products. Certain 5′ nuclease probes include labeling moieties, such as a fluorescent reporter dye and a quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye generally results in the suppression of the reporter fluorescence. In many cases, however, an intact probe produces a certain amount of residual or baseline fluorescence. During a 5′ nuclease reaction, cleavage of the probe separates the reporter dye and the quencher dye from one another, resulting in a detectable increase in fluorescence from the reporter. The accumulation of PCR products or amplicons is typically detected indirectly by monitoring this increase in fluorescence in real-time.